1. Field of the Invention
The present invention relates to a method for optically detecting at least one entity on or in a substrate, preferably arranged on a support. Furthermore, fields of application of the method according to the invention as well as a device for carrying out said method are described.
2. Description of Related Art
It is known that confocal arrangements or arrangements being constructed for multi-photons-excitation due to their high axial local-resolution are suitable for reduction of background signals which are outside of the focal plane. Thus, in particular when detecting large-surface-structures, there is the problem that during the scanning process it has to be ensured that the focal plane is always situated in a desired position within the objects to be examined. Therefore it is possible that, for example, irregularities of a sample-support, the object to be examined is arranged on, lead to that the confocal measuring volume is not in the desired plane within the object but possibly detects structures adjacent said object, as for example parts of the sample-support. This adversely effects the object's registration and characterization that has to be performed. Therefore it is desired to take measures to maintain or to track the focal plane within a certain position.
“Patent Abstracts of Japan” (vol. 018, no. 436 (P-1786), Aug. 15, 1994) describes a device for detecting the focus-position suitable for automatically focussing an image-generating device or for measuring inequalities of the surface of an object to be examined. An optical fiber is used the end of which is moved along an optical axis by means of an actuator. The so produced interfering signal is used for the detection of deviation of the focus-position as well as for readjustment of the same.
“Patent Abstracts of Japan” (vol. 098, no. 004, Mar. 31, 1998) proposes a focus-detector using the principle of confocal microscopic optics. U.S. Pat. No. 5,062,715 discloses the use of a confocal autofocus system in a Michelson-interferometer designed for the measurement of surface-vibration.
U.S. Pat. No. 5,084,612 describes an image generating method for a scanning-microscope constructed in transmission-geometry. Herein, the position of an apertured diaphragm used for detection is tracked in a manner that possible deviation of transmitted light occurring in proximity of (in the section of?) the sample because of refraction-effects are compensated. However, it is not the object of said method to track the position of the measuring focus within the sample.
PCT/US95/01886 (international publication number WO 95/22058) describes a confocal detection device having an automatic focussing mechanism including a confocal apertured diaphragm. The autofocusing is realized in three steps. First, a laser is focused on the back side of a substrate having the sample applied thereon. In a further step the focus is positioned in a plane above the substrate. It is only in a third step, that after passing the desired position on the surface of said substrate the exact position of the surface is determined and the focus is adjusted on the substrate-surface. This process is performed at the four corners of the substrate which is an extremely time-consuming procedure. It is not possible to operate the autofocussing system during the actual measurement of the sample and the focus-height is estimated by interpolation. Hereby, not acceptable positioning defaults may occur, in particular with substrates which are not plan, as they are normally used in laboratories for cost reasons.